Proliferating Cell Nuclear Antigen Promotes DNA Trinucleotide Repeat Hairpin Removal

Background: Endogenous and environmental stress lead to the formation of DNA base lesions, which are repaired in cells through DNA base excision repair (BER). BER of DNA base lesions within trinucleotide repeat (TNR) tracts can result in repeat instability in the form of both expansions and deletions, which are linked to over 40 human neurodegenerative diseases and cancers. This instability results from the formation of non-B form DNA secondary structures and the resulting loss of coordination between BER enzymes. Inefficient flap endonuclease 1 (FEN1) 5’-flap cleavage during BER leads to TNR expansions, and repeated cycles of inefficient BER result in cumulative TNR expansions and the onset of disease. However, the molecular mechanisms underlying environmentally induced TNR instability and the mechanisms by which cells prevent TNR expansion remain to be elucidated. Determining the role of BER proteins and cofactors in modulating TNR instability can aid in the identification of novel targets for the therapeutic treatment of this family of diseases. Proliferating cell nuclear antigen (PCNA) is a cofactor that plays a crucial role during DNA replication and BER by stimulating the flap cleavage activity of FEN1. However, it is unknown whether PCNA has any stimulatory effects on FEN1 flap cleavage of a TNR flap to prevent TNR expansion during BER. Methods: We explored the effects of PCNA on CAG repeat stability and FEN1 cleavage activity during BER of a lesion located within a duplex CAG-repeat tract, as well as within the loop region of a CAG-repeat hairpin, through reconstitution of BER and in vitro enzymatic assays using synthesized DNA oligonucleotides. Results: We found that PCNA stimulated FEN1 flap cleavage activity during BER of a lesion located in a hairpin loop, but failed to affect FEN1 flap cleavage activity during BER in a TNR duplex. We demonstrated that PCNA promoted efficient BER and the formation of unexpanded repaired products during BER of a lesion located in a hairpin loop by facilitating removal of the TNR hairpin. Conclusions: We conclude that the effects of PCNA on TNR stability are highly dependent on the location of a lesion, with the structure of a TNR tract governing the outcome of the PCNA-FEN1 interaction. While PCNA was unable to facilitate efficient repair within a TNR duplex, PCNA does stimulate TNR hairpin removal, preventing or attenuating TNR expansions during BER in a TNR hairpin loop.